DESCRIPTION (Investigator's Abstract): The specific goal of this proposal for competing renewal is to continue our efforts towards unfolding some of the intricacies of the mechanistic relationship between increased phosphoinositide (PI) hydrolysis and activation of neuronal nitric oxide (NO) synthase mediated by muscarinic receptors. The understanding of this relationship is of paramount importance due to the prominent role of both signaling systems in the regulation of neuronal function. our findings during the current grant period demonstrate that different subtypes of muscarinic receptors vary markedly in the efficiency of their coupling to activation of neuronal NO synthase. Unexpectedly, we have obtained multiple lines of evidence which strongly suggest that cellular mechanisms which are linked to PI hydrolysis and others which might be independent of this pathway both contribute to muscarinic receptor mediated increase in NO release, and that various receptor subtypes might utilize each of the two pathways to a different extent. Alternatively, we hypothesize that muscarinic receptors which are efficiently coupled to stimulation of NO generation might utilize more than one cellular source of calcium to activate NO synthase. Last, we obtained very exciting data indicating that No is able to feed back to regulate muscarinic receptor-mediated activation of NO synthase and stimulation of PI hydrolysis in a negative manner. As a result of our findings, we plan to study the following focused, inter-related and hypothesis driven Specific Aims in the next funding period: (a) examine the selectivity of coupling of m1-m5 muscarinic receptors to activation of neuronal NO synthase, and determine the relative dependence of this signaling mechanism on PI hydrolysis at the various receptor subtypes; (b) delineate the relative role of Ca2+ mobilization from intracellular stores and its influx through the cell membrane in coupling of different muscarinic receptor subtypes to activation of neuronal NO synthase, and examine the dependence of the two pools of Ca2+ on PI hydrolysis; (c) investigate the role of generated NO in feedback modulation of coupling of muscarinic receptors to stimulation of signaling mechanisms which result in activation of NO synthase. The planned studies represent a logical extension of the goals of the current grant and are expected to result in significant advances in our understanding of the bidirectional cross-talk between two of the most important second messenger signaling mechanisms namely PI hydrolysis and NO.